The use of selectable marker genes, such as the kanamycin resistance encoding neomycin phosphotransferase (nptII), has been invaluable in transgenic plant production. Since all currently used antibiotic resistance genes are from bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants to microbes thereby causing new antibiotic resistance problems. Here we characterize the first, to our knowledge, plant gene shown to confer antibiotic resistance in transgenic plants: an Arabidopsis thaliana ATP binding cassette (ABC) transporter, Atwbc19. Its mechanism of conferring resistance is novel while the level of endowed resistance is comparable to that of bacterial antibiotic resistance genes in transgenic tobacco using the 35S promoter. Thus, since ABC transporters are endogenous to plants, there should be less controversy using Atwbc19, as a selectable marker in transgenic plants with regards to concerns of horizontal gene transfer.
ABC proteins are biologically ubiquitous proteins classified on the basis of the presence of an ATP-binding cassette or nucleotide binding folds, with sharing of 30-40% identity between family members (Higgins, 1992). The vast majority of ABC-transporters are membrane bound and contain transmembrane domains (TMDs). These TMDs are considered to form the pathway for solute movement across the phospholipid bilayer and appear to determine, or at least contribute to, the substrate selectivity of the transporter. Resistance of human cancer cells to chemotherapeutic agents and multidrug resistance in infectious microorganisms often arises from the over-expression of ABC transporters. Their clinical significance has spurred a number of structural studies to better understand how ATP hydrolysis is coupled with a substrate specific transport.
Among all multicellular organisms sequenced so far, plants have the largest number of ABC proteins encoded in their genome. Why plants allocate proportionally more genes to this superfamily relative to their genome size is not clear, but begs the question of adaptive significance (Sanchez-Fernandez et al., 2001a). Arabidopsis ABC transporters have been classified on the basis of their domain organization and their homology to orthologous genes, but their functions remain largely unknown (Sanchez-Fernandez et al., 2001b). The AtWBC family (White-Brown Complex homologues) is the largest of all Arabidopsis ABC transporters with 29 members. One recent publication attributes the role of Atwbc12 to the secretion of cuticular wax (Pighin et al., 2004).
Kanamycin is an aminoglycoside antibiotic isolated from the soil bacterium Streptomyces kanamyceticus. Aminoglycosides act primarily by binding to the 30S subunit of prokaryotic ribosomes and inhibiting protein synthesis (Mingeot-Leclercq et al., 1999). In eukaryotes they inhibit protein synthesis by putative non-specific binding to the ribosomal complex, hence their usefulness as selection agents for plant and mammalian genetic transformation. In bacteria, aminoglycoside resistance is most often associated with the presence of inactivating enzymes. These enzymes, classified as aminoglycoside acetyltransferases, nucleotidyltransferases or phosphotransferases, catalyze the transfer of acetyl, adenosine monophosphate, or phosphate groups onto the aminoglycoside antibiotics (Wright et al., 1998). Neomycin phosphotransferase type II (nptII), originally isolated from the Tn5 transposon of Escherichia coli, is among enzymes of the latter group. Since its first use in 1983 (Bevan et al., 1983; Fraley et al., 1983; Herrera-Estrella et al., 1983), it has been the most commonly used selectable marker for the production of transgenic plants. The fact that kanamycin resistance via aminoglycoside modifying enzymes is common among soil bacteria has contributed to its acceptance by regulatory agencies for deregulated transgenic plants containing nptII.